Program Director/Principal Investigator (Last, First, Middle): Mann, Barbara J. 1 R21 AI073385-01A1 ABSTRACT Francisella tularensis is a highly efficient zoonotic pathogen that is able to establish a virulent infection with 10 organisms or less. To accomplish this feat the bacterium has devised efficient means of infecting its host, and effective ways of avoiding host defense mechanisms. Few virulence factors or virulence-associated genes have been identified, and it has no known toxins. To identify virulence factors in F. tularensis a transposonmutagenesis library of the Schu S4 strain of F. tularensis was constructed. Schu S4 is a human isolate that belongs to the virulent subspecies tularensis, and is considered a Category A Select Agent of biological warfare. The library was screened for mutants defective in intracellular survival in the hepatic cell line HepG2. This screen identified a dsbB mutant. dsbB encodes the inner membrane disulfide bond formation protein B that oxidizes disulfide bond formation protein A (DsbA) to its active form. DsbA in turn catalyzes disulfide bond formation in proteins that are in, or transit through, the periplasm. A dsbB mutant is intriguing because it is necessary for the formation of critical virulence factors such as type III secretion and type IV pili biogenesis in other bacteria. We hypothesize that the DsbA/DsbB pathway is critical for the formation of virulence factors in Francisella. Understanding the role of the DsbA/DsbB pathway in intracellular survival may be a means to identify potential virulence factors, and increase the understanding of the mechanisms that F. tularensis uses to adapt to the host milieu. To identify the substrates of DsbA/DsbB pathway protein profiles of the dsbB mutants will be compared to wild-type bacteria using polyacrylamide gels and mass spectrometry techniques. Several substrates will be selected for further study. The role that these substrates play in intracellular survival and virulence will be characterized genetically, biochemically, and in vivo. Identification of novel virulence factors may have practical applications for the development of attenuated vaccines, and/or the identification of targets for new therapeutics.